Serine proteases are a class of proteins that proteolytically cleave other proteins. Members of this class of proteins contribute to important biological processes including the proteolytic cascade reactions of complement activation and blood coagulation.
Cleavage of a blood coagulation factor contributes to the coagulation cascade, resulting in blood coagulation. A variety of medical conditions can arise where it is advantageous to inhibit the coagulation cascade at the level of one or another proteolytic step. In addition, procedures involving blood product manipulation can activate members of the cascade, and therefore their specific inhibition is advantageous.
Protein C (PC) is a member of the class of vitamin K-dependent serine protease coagulation factors. Unlike the majority of coagulation factors, such as Factors VIIa, IXa, Xa, XIIa, thrombin, plasmin or plasma kallikrein which are procoagulants, Protein C regulates blood coagulation by acting as a natural anticoagulant that circulates in the blood in an inactive form that requires proteolytic activation to generate the anticoagulant enzyme. The activated form of Protein C, APC, inhibits blood coagulation at the levels of Factor V and VIII in the clotting cascade.
Similar to most other zymogens of extracellular proteases and the above recited blood coagulation factors, Protein C has the core structure of the chymotrypsin family having insertions and N-terminus extensions that enable regulation of the zymogen and the enzyme. See Owen W., in "Hemostasis and Thrombosis: Basic Principles and Clinical Practice", Colman et al., eds, pp. 235-241, J.B. Lippincott Co. (Philadelphia), 1987.
Protein C is composed of domains with discrete structure and function. See Foster et al., Proc. Natl. Acad. Sci. USA. 82:4673-4677 (1985) and Plutzky et al., Proc. Natl. Acad. Sci. USA, 83:546-550 (1986). The light chain contains an amino-terminal gamma-carboxyglutamic acid (Gla) region which is followed by two domains that are homologous to domains in the epidermal growth factor (EGF) precursor. The serine protease activity resides in the heavy chain. Ohlin et al., Biochem., 29:644-651 (1990).
The zymogen is activated by the action of thrombin at the site between the arginine residue at position number 12 and the leucine residue at position 13. See Kisiel, J. Clin. Invest., 64:761-769, (1976); Marlar et al., Blood, 59:1067-1072 (1982). Other proteins including Factor Xa (Haley et al., J. Biol. Chem., 264:16303-16310 (1989), Russell's viper venom and trypsin (Esmon et al., J. Biol. Chem., 251:2770-2776 (1976) have also been shown to enzymatically cleave inactive protein C to its activated form. Activated protein C hydrolyzes arginine esters and related substrates via a core triad of catalytic amino acid residues which occur at Ser-201, His-56, and Asp-102 of the heavy chain. The triad forms a hydrophobic substrate binding pocket. The enzyme's specificity is restricted to a small number of protein substrates; blood coagulation cofactors, activated Factors V and VIII, are the only known macromolecular substrates for the proteolytic inactivation by activated protein C. See Kisiel et al., Biochem., 16:5824-5831 (1977); Vehar et al., Biochem., 19:401-410 (1980); and Walker et al., Biochim. Biophys. Acta., 571:333-342 (1979).
Thrombin, the major physiological protein C activator, activates protein C slowly in purified systems, plasma, or blood, when in the presence of physiological concentrations of calcium. A membrane-bound thrombin receptor called thrombomodulin has been identified which accelerates protein C activation. Esmon et al., Proc. Natl. Acad. Sci. USA, 78:2249-2252 (1981). Liberated thrombin binds to thrombomodulin on the luminal surface of endothelial cells and undergoes an increase in specificity for circulating protein C. Calcium is required for this process and is bound to calcium-binding domains in the EGF-like regions of protein C. Additional studies have revealed that the membrane-lipid domain of protein C, the vitamin-K dependent Gla domain, is also required for activation of protein C. Esmon et al., in "Progress in Vascular Biology, Hemostasis, and Thrombosis", Ruggeri et al., eds., Annals of The New York Academy of Sciences, Vol. 614:30-43 (1991).
Inhibitors of members of the coagulation serine proteases have been described. For example, a region of Factor VIIa comprising residues 285 to 305 was reported to inhibit the Factor VIIa-tissue factor mediated conversion of Factor X to Factor Xa. Kumar et al, J. Biol. Chem., 266:915-921 (1991). In addition, a synthetic polypeptide corresponding to the region of prothrombin at residues 467 to 478 was reported to inhibit binding of thrombin to thrombomodulin and to inhibit thrombin induced clotting of fibrinogen. Suzuki et al, Blood, 77:317-323 (1991).